24-hydroxyvitamin D, analogs and uses thereof

ABSTRACT

The invention provides 24-hydroxyvitamin D compounds and methods for their use in the treatment and prophylaxis of hyperparathyroidism and hyperproliferative diseases, and in the modulation of the immune and inflammatory responses as well as the treatment of bone depletive disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/086,969, filed May, 29, 1998, now U.S. Pat. No. 6,242,434.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

This invention relates generally to 24-hydroxyvitamin D compounds andtheir use in the treatment and prophylaxis of hyperparathyroidism andhyperproliferative diseases, and in the modulation of the immuneresponse as well as the treatment of bone depletive disorders.

Vitamin D has long been established as having an important biologicalrole in bone and mineral metabolism. For example, vitamin D plays acritical role in stimulating calcium absorption and regulating calciummetabolism. The discovery of active forms of vitamin D, (M. F. Holick etal., 68 Proc. Natl. Acad. Sci. USA, 803–804 (1971); G. Jones et al., 14Biochemistry, 1250–1256 (1975)), and active vitamin D analogs (M. F.Holick et al., 180 Science 190–191 (1973); H. Y. Lam et al., 186 Science1038–1040 (1974)) caused much excitement and speculation about theusefulness of these vitamin D compounds in the treatment of bonedepletive disorders.

Animal studies examining the effects of these active vitamin D compoundssuggested that such agents would be useful in restoring calcium balance.An early clinical study indicated that administration of 0.5 μg/day of1α,25-dihydroxyvitamin D₃, the hormonally active form of vitamin D₃, toa group of postmenopausal women improved intestinal calcium absorptionas well as calcium balance in the women. On this basis, U.S. Pat. No.4,225,596 (“'596 Patent”) described and claimed the use of1α,25-dihydroxyvitamin D₃ and analogs thereof for increasing calciumabsorption and retention.

The best indicator of the efficacy of vitamin D compounds in theprevention or treatment of depletive bone disorders, however, is boneitself rather than calcium absorption or calcium balance. More recentclinical data indicate that, at the dosage ranges taught in the '596Patent, 1α,25-dihydroxyvitamin D₃ has, at best, modest efficacy inpreventing or restoring loss of bone mass or bone mineral content (S. M.Ott and C. H. Chesnut, 110 Ann. Int. Med. 267–274 (1989); J. C.Gallagher et al., 113 Ann. Int. Med. 649–655 (1990); J. Aloia et al., 84Amer. J. Med. 401–408 (1988)).

These clinical studies with 1α,25-dihydroxyvitamin D₃, and anotherconducted with 1α-hydroxyvitamin D₃ (M. Shiraki et al., 32 Endocrinol.Japan 305–315 (1985)), indicate that the capacity of these two vitamin Dcompounds to restore lost bone mass or bone mineral content isdose-related. The studies also indicate, however, that, at the dosageranges required for either compound to be truly effective, toxicity inthe form of hypercalcemia and hypercalciuria becomes a major problem.Specifically, attempts to increase the amount of 1α,25-dihydroxyvitaminD₃ above 0.5 μg/day have frequently resulted in toxicity. At dosagelevels below 0.5 μg/day, no effects are observed on bone mass or mineralcontent. (See, G. F. Jensen et al., 16 Clin. Endocrinol. 515–524 (1982);C. Christiansen et al., 11 Eur. J. Clin. Invest. 305–309 (1981)).

Data from clinical studies in Japan, a population that has low calciumintake, indicate that efficacy is found with 1α-hydroxyvitamin D₃ whenadministered at 1 μg/day (M. Shiraki et al., 32 Endocrinol. Japan.305–315 (1985); H. Orimo et al., 3 Bone and Mineral 47–52 (1987)). Twoμg/day of 1α-hydroxyvitamin D₃ was found to have efficacy in increasingbone mass in patients exhibiting senile osteoporosis (O. H. Sorensen etal., 7 Clin. Endocrinol. 169S–175S (1977)). At 2 μg/day, however,toxicity with 1α-hydroxyvitamin D₃ occurs in approximately 67 percent ofthe patients, and at 1 μg/day, this percentage is approximately 20percent. Thus, these 1α-hydroxylated vitamin D₃ compounds can producedangerously elevated blood calcium levels due to their inherent calcemicactivity.

Because of this toxicity, 1-hydroxylated vitamin D₃ compounds can onlybe administered at dosages that are, at best, modestly beneficial inpreventing or treating loss of bone or bone mineral content. Indeed,Aloia recommends that alternative routes of administration be soughtwhich might avoid the toxicity problems and allow higher dosage levelsto be achieved. (J. Aloia et al., 84 Amer. J. Med. 401–408 (1988).) Yet,despite reported toxicities of 1α-hydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃, these two compounds remain the drugs ofchoice for many bone depletive disease treatments.

These two drugs also remain the only approved forms of 1α-hydroxylatedvitamin D for treating or preventing hyperparathyroidism which occurssecondary to end stage renal disease, although both drugs are notcurrently approved in all major pharmaceutical markets.Hyperparathyroidism is a generalized disorder resulting from excessivesecretion of parathyroid hormone (PTH) by one or more parathyroidglands. It is thus characterized by elevated blood levels of parathyroidhormone. Typically, one or more parathyroid glands reveal a markedenlargement. In the case of primary hyperparathyroidism, the glandularenlargement is usually due to a neoplasm or tumor. In the case ofsecondary hyperparathyroidism, the parathyroid gland hyperplasiatypically occurs because of resistance to the metabolic actions of thehormone. Secondary hyperparathyroidism occurs in patients with, e.g.,renal failure, osteomalacia, and intestinal malabsorption syndrome. Inboth primary and secondary hyperparathyroidism, bone abnormalities,e.g., the loss of bone mass or decreased mineral content, are common andrenal damage is possible. Hyperparathyroidism is thus also characterizedby abnormal calcium, phosphorus and bone metabolism.

More recently, other roles for vitamin D have come to light. Specificnuclear receptors for 1α,25-dihydroxyvitamin D₃ have been found in cellsfrom diverse organs not involved in calcium homeostasis. For example,Miller et al., 52 Cancer Res. (1992) 515–520, have demonstratedbiologically active, specific receptors for 1α,25-dihydroxyvitamin D₃ inthe human prostatic carcinoma cell line, LNCaP.

It has been reported that certain vitamin D compounds and analogs arepotent inhibitors of malignant cell proliferation andinducers/stimulators of cell differentiation. For example, U.S. Pat. No.4,391,802 issued to Suda et al. discloses that 1α-hydroxyvitamin Dcompounds, specifically, 1α,25-dihydroxyvitamin D₃ and 1α-hydroxyvitaminD₃, possess potent antileukemic activity by virtue of inducing thedifferentiation of malignant cells (specifically, leukemia cells) tononmalignant macrophages (monocytes), and are useful in the treatment ofleukemia. In another example, Skowronski et al., 136 Endocrinology 20–26(1995), have reported antiproliferative and differentiating actions of1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogs on prostatecancer cell lines.

Previous proliferation studies, such as those cited above, focusedexclusively on vitamin D₃ compounds. Even though such compounds may,indeed, be highly effective in differentiating malignant cells inculture, their practical use in differentiation therapy as anticanceragents is severely limited because of their equally high potency asagents affecting calcium metabolism. At the levels required in vivo foreffective use as antileukemic agents, these same compounds can inducemarkedly elevated and potentially dangerous blood calcium levels byvirtue of their inherent calcemic activity. In other words, the clinicaluse of 1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogs asanticancer agents is precluded, or severely limited, by the risk ofhypercalcemia.

Still other metabolic conditions in which it has been suggested thatvitamin D plays a role are immune response (see, e.g., U.S. Pat. No.4,749,710 issued to Truitt et al.; U.S. Pat. No. 5,559,107 issued toGates et al.; U.S. Pat. Nos. 5,540,919, 5,518,725 and 5,562,910 issuedto Daynes et al.) and inflammatory response (e.g., U.S. Pat. No.5,589,471 issued to Hansen et al.).

Considering the diverse biological actions of vitamin D and itspotential as a therapeutic agent, a need exists for compounds withgreater specific activity and selectivity of action, e.g., vitamin Dcompounds with antiproliferative and differentiating effects but whichhave less calcemic activity than therapeutic amounts of the knowncompounds or analogs of vitamin D₃.

BRIEF SUMMARY OF THE INVENTION

The present invention provides 24-hydroxyvitamin D compounds representedby general formula (I) described hereinafter, wherein the C-17 sidechainis a saturated or unsaturated, substituted or unsubstituted, straight orbranched C₄–C₁₈ hydrocarbon group in which the C-24 or equivalentposition is hydroxylated. The invention also provides a method fortreating or preventing certain diseases and disorders. Such diseases anddisorders include (i) hyperparathyroidism by lowering (or maintaininglow) serum parathyroid hormone levels; (ii) hyperproliferative diseases;(iii) immune response imbalance; (iv) inflammatory diseases; and (v)bone depletive disorders.

The compound of formula (I) is a 24-hydroxyvitamin D compound which haspotent biological activity but low calcemic activity relative to theactive forms of vitamin D₃. Preferably such compounds are24-hydroxylated prodrugs which are hydroxylated in vivo at the C-1position to form 1,24-dihydroxylated active vitamin D compounds.

As used herein, the term “vitamin D compound” is meant to refer tocompounds which fall within the generic structure of formula (I) andsuch compound or its metabolite exhibits vitamin D hormonal bioactivity.It is also noted that a shorthand notation is often used for the Dcompounds, e.g., 1α-hydroxyvitamin D₂ may be referred to as simply1α-OH-D₂.

In another aspect, the invention is a pharmaceutical composition inwhich the compound of formula (I) is the active ingredient. Suchcomposition is suitably presented in unit dosage form having aneffective amount of a vitamin D compound of formula (I) and apharmaceutically acceptable excipient.

The treatment methods of the present invention are alternatives toconventional therapies with 1α,25-dihydroxyvitamin D₃ or1α-hydroxyvitamin D₃. The methods are characterized as providing thecompound of formula (I) having equivalent bioactivity but much lowertoxicity than these conventional therapies.

Other advantages and a fuller appreciation of the specific attributes ofthis invention will be gained upon an examination of the followingdrawings, detailed description of preferred embodiments, and appendedclaims. It is expressly understood that the drawings are for the purposeof illustration and description only, and are not intended as adefinition of the limits of the invention.

BRIEF DESCRIPTION OF THE DRAWING(S)

The preferred exemplary embodiment of the present invention willhereinafter be described in conjunction with the appended drawingwherein like designations refer to like elements throughout and inwhich:

FIG. 1 is an exemplary reaction scheme for the preparation of24-hydroxyvitamin D₂;

FIG. 2 is an exemplary reaction scheme for the preparation of24-hydroxy-25-ene-vitamin D₂;

FIG. 3 is an IR spectrum of 24(S)-hydroxyvitamin D₂; and

FIG. 4 is an NMR spectrum of the compound of FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates 24-hydroxyvitamin D compounds. Thecompounds of the present invention are most particularly adapted for usein the treatment and prophylaxis of certain diseases and disorders,e.g., hyperproliferative and inflammatory diseases, hyperparathyroidism,bone depletive disorders and certain immune response conditions. Suchhyperproliferative diseases include skin, breast, colon and prostatecancer and psoriasis. Inflammatory diseases include arthritis andasthma. Hyperparathyroid diseases include both primary and secondaryhyperparathyroidism. Immune response conditions include autoimmunediabetes, multiple sclerosis and transplant rejection. Accordingly, thepresent invention will now be described in detail with respect to suchendeavors; however, those skilled in the art will appreciate that such adescription of the invention is meant to be exemplary only and shouldnot be viewed as limitative on the full scope thereof.

The present invention provides 24-hydroxyvitamin D compounds which findvalue as pharmaceutical agents. These compounds are suitably prodrugsfor 1α,24-dihydroxylated vitamin D compound as they are hydroxylated invivo at the 1α-position to become active forms of vitamin D. Asprodrugs, these compounds, in effect, by-pass the first-pass concernover intestinal vitamin D receptor binding which mediates intestinalcalcium absorption, thereby resulting in reduced or no hypercalcemiacompared with similar dosing with known active vitamin D compounds suchas 1α,25-dihydroxyvitamin D₃ and 1α-hydroxyvitamin D₃.

In the following description of the method of the invention, processsteps are carried out at room temperature and atmospheric pressureunless otherwise specified.

As used herein, the terms “substantially pure” or “substantially free”refer to a purity of at least 90%. The term “substantially less” refersto at least 25% less than the comparative substance. Also, as usedherein, the term “lower” as a modifier for alkyl, alkenyl, fluoroalkyl,fluoroalkenyl or cycloalkyl is meant to refer to a straight or branched,saturated or unsaturated hydrocarbon group having 1 to 4 carbon atoms.Specific examples of such hydrocarbon groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl,isobutenyl, isopropenyl, formyl, acetyl, propionyl, butyryl orcyclopropyl. As used herein, the term “hydrocarbon moiety” refers to alower alkyl, a lower alkenyl, a lower acyl group or a lower cycloalkyl,i.e., a straight or branched, saturated or unsaturated C₁–C₄ hydrocarbongroup. Also, the term “equivalent position,” as in, e.g., C-24 orequivalent position, is meant to refer to a particular carbon in theC-17 side chain of a vitamin D compound wherein that carbon would be theC-24 carbon but for homologation of the side chain.

In one aspect, vitamin D compounds operable in the present invention aresuitably represented by formula (I):

wherein Z represents a saturated or unsaturated, substituted orunsubstituted, straight-chain or branched C₄–C₁₈ hydrocarbon group inwhich the C-24 or equivalent position is hydroxylated; Y is a methylenegroup if Y is double bonded to the A-ring or a methyl or hydrogen if Yis single bonded, i.e., when Y is hydrogen, the compound of formula (I)is a 19-nor compound; and X is hydrogen, lower alkyl or lowerfluoroalkyl. It is noted that when X is hydrogen, the compound offormula (I) is an 18-nor compound.

Preferably, Z is a side chain represented by formula (IIA):

wherein m is 0 or 1 and a dotted line along the side chain represents anoptional additional C—C bond; R¹ and R² are independently lower alkyl,lower fluoroalkyl, lower alkenyl, lower fluoroalkenyl, lower cycloalkylor, taken together with the carbon to which they are bonded (e.g., C-25in the case where m=0), form a C₃–C₈ cyclohydrocarbon ring; R³ ishydrogen, lower alkyl, lower fluoroalkyl, lower alkenyl or lowerfluoroalkenyl; R⁴ is lower alkyl, lower fluoroalkyl, lower alkenyl orlower fluoroalkenyl; R⁵ and R⁶ are each hydrogen or taken together forma double bond between C-22 and C-23. As to the bond to which m refers,this bond may be a single, double or triple bond, in other words, a—CH₂—CH₂—, a —CH═CH— or a —C≡C—.

For example, Z includes a side chain represented by formula (IIB):

wherein R⁵ and R⁶ are each hydrogen or taken together form a double bondbetween C-22 and C-23, R³ is hydrogen, lower alkyl, lower fluoroalkyl,lower alkenyl or lower fluoroalkenyl; R⁴ is lower alkyl, lowerfluoroalkyl, lower alkenyl or lower fluoroalkenyl; and R¹ and R² areindependently hydrogen, lower alkyl, lower fluoroalkyl, lower alkenyl,lower fluoroalkenyl, lower cycloalkyl or taken together with the carbonto which they are bonded (i.e., C-25) form a C₃–C₈ cyclocarbon ring.

Z also includes a side chain represented by formula (IIC):

wherein n is an integer which is 1 or 2; R³ is hydrogen, lower alkyl,lower fluoroalkyl, lower alkenyl or lower fluoroalkenyl; R⁴ and R⁷ areindependently lower alkyl, lower fluoroalkyl, lower alkenyl or lowerfluoroalkenyl; A is carbon, oxygen, sulfur or nitrogen; r is 1 and s iszero when A is nitrogen; r and s are 1 when A is carbon; r and s arezero when A is sulfur or oxygen; and R⁹ and R¹⁰ are independentlyhydrogen, lower alkyl, lower alkenyl, lower fluoroalkyl or lowerfluoroalkenyl. As to the bond to which m refers, this bond is a—CH₂—CH₂— or a —CH═CH—.

For example, Z includes a side chain wherein n is 1, A is carbon and rand s are 1 and which is represented by formula (IID):

wherein R³, R⁹ and R¹⁰ are independently hydrogen, lower alkyl, lowerfluoroalkyl, lower alkenyl or lower fluoroalkenyl, and R⁴ and R⁷ areindependently lower alkyl, lower fluoroalkyl, lower alkenyl or lowerfluoroalkenyl.

Also, Z includes a side chain represented by formula (IIE):

wherein a dotted line along the side chain represents an optionaladditional C—C bond; q is zero or an integer which is 1 or 2; R³ ishydrogen, lower alkyl, lower fluoroalkyl, lower alkenyl or lowerfluoroalkenyl; R⁴ and R⁷ are independently lower alkyl, lowerfluoroalkyl, lower alkenyl or lower fluoroalkenyl; A is carbon, oxygen,sulfur or nitrogen; r is 1 and s is zero when A is nitrogen; r and s are1 when A is carbon; r and s are zero when A is sulfur or oxygen; R⁹ andR¹⁰ are independently hydrogen, lower alkyl, lower alkenyl, lowerfluoroalkyl or lower fluoroalkenyl. As to the optional additional bonds,for example, when q=0, there may be a single, double or triple bondbetween C-22 and C-23. As to the group to which q refers, this group is—CH₂—.

For example, Z includes a side chain wherein q is zero, A is carbon, rand s are 1; R³, R⁹ and R¹⁰ are hydrogen, lower alkyl, lowerfluoroalkyl, lower alkenyl or lower fluoroalkenyl; R⁴ and R⁷ areindependently lower alkyl, lower fluoroalkyl, lower alkenyl or lowerfluoroalkenyl; and which is represented by formula (IIF):

Also included as vitamin D compounds within the scope of the presentinvention are 24-hydroxyprevitamin D compounds which suitably includethe same Z side chain described above including those represented byformulas (IIA), (IIB), (IIC), (IID), (IIE) and (IIF) that arehydroxysubstituted at C-24 or equivalent position. Previtamin Dcompounds are the thermal isomers of the corresponding vitamin Dcompounds, e.g., 24-hydroxyprevitamin D₂ is the thermal isomer of24-hydroxyvitamin D₂, and exists in thermal equilibrium with same.24-Hydroxyprevitamin D compounds in accordance with the presentinvention are suitably represented by formula (III):

wherein X, Y and Z are as described above.

Preferred among the compounds of formula (I) are the 24-hydroxycompounds which are prodrugs for 1α,24-dihydroxylated vitamin D.Examples of the compounds of formula (I) are:

24-hydroxyvitamin D₂ [24-(OH)-D₂];

24-hydroxy-25-fluorovitamin D₂ [24-(OH)-25-F-D₂];

24-hydroxy-25-ene-vitamin D₂ [24-OH-25-ene-D₂];

24-hydroxy-25-oxo-vitamin D₂ [24-OH-25-oxo-D₂];

24-hydroxyvitamin D₄ [24-(OH)-D_(4];)

24-hydroxy-25-fluorovitamin D₄[24-(OH)-25-F-D₄];

24-hydroxy-25-ene-vitamin D₄ [24-OH-25-ene-D₄]; and

24-hydroxy-25-oxo-vitamin D₄ [24-OH-25-oxo-D₄].

Preferred among the compounds of formula (III) are the 24-hydroxyprevitamin D compounds which are prodrugs and isomers for1α,24-dihydroxylated vitamin D. Examples of the compounds of formula(III) are:

24-hydroxyprevitamin D₂ [24-(OH)-preD₂];

24-hydroxy-25-fluoroprevitamin D₂ [24-(OH)-25-F-preD₂];

24-hydroxy-25-ene-previtamin D₂ [24-OH-25-ene-preD₂];

24-hydroxy-25-oxo-previtamin D₂ [24-OH-25-oxo-preD₂];

24-hydroxyprevitamin D₄ [24-(OH)-preD₄];

24-hydroxy-25-fluoroprevitamin D₄ [24-(OH)-25-F-preD₄];

24-hydroxy-25-ene-previtamin D₄ [24-OH-25-ene-preD₄]; and

24-hydroxy-25-oxo-previtamin D₄ [24-OH-25-oxo-preD₄].

Among those compounds of the present invention that have a chiral centerin the sidechain, such as at C-20 or C-24, it is understood that bothdiastereomers (e.g., R and S) and the mixture thereof are within thescope of the present invention.

The compounds of formula (I) may generally be prepared by the exemplaryreaction process depicted in FIG. 1. FIG. 1 illustrates a method ofpreparing 24-hydroxyvitamin D₂ using ergosterol as a starting materialand forming 24-hydroxyvitamin D₂ which is then separated to yield the24(S)-hydroxyvitamin D₂ diastereomer and the 24(R)-hydroxyvitamin D₂diastereomer, if stereochemical purity is desired. Hereinafter whenreference is made to a 24-hydroxy compound, unless otherwise specified,it will be presumed that the compound is a diastereomeric mixture of theR and S forms.

Specifically, ergosterol is converted to 24-hydroxyergosterol (5,7,22ergostatriene-3β,24-diol (7)) by a six-step process. The24-hydroxyergosterol is then irradiated and thermally converted bymethods well known in the art to yield 24-hydroxyvitamin D₂ from whichthe diastereomers are separated.

As seen in FIG. 1, ergosterol is acetylated to form the 3β-acetate (2).An adduct (3) is then formed with the B-ring of the ergosterol structureby reaction of the 3β-acetate with a triazoline dione. The adduct (3) isthen ozonated to truncate the side chain to form a C-21 aldehyde (4).The side chain is reestablished by reaction of the resulting aldehydewith the appropriate keto-compound to yield the 24-enone (5). The enoneis then converted to the 24-methyl, 3β,24-dihydroxy adduct (6). Thisadduct is then reacted with a lithium aluminum hydride to deprotect theadduct and yield 24-hydroxyergosterol (7). The 24-hydroxyergosterol isthen irradiated and thermally treated to form 24-hydroxyvitamin D₂ (8).The 24-hydroxyvitamin D₂ is subjected to reverse phase high pressureliquid chromatography to separate the two diastereomers and recover thediastereomeric forms of the invention, 24(S)-hydroxyvitamin D₂ (9) and24(R)-hydroxyvitamin D₂ (10).

The compounds of formula (I) wherein the side chain is represented byformulas (IIC) or (IIE) may generally be prepared by the exemplaryreaction process depicted in FIG. 2. FIG. 2 illustrates a method ofpreparing 24-hydroxy-25-ene-vitamin D₂ entails using vitamin D₂ as astarting material and forming 25-ene-vitamin D₂, incubating culturedcells derived from human hepatoma cells, e.g., HEP3B or HEPG2, with the25-ene-vitamin D₂ to yield the metabolite 24-hydroxy-25-ene-vitamin D₂which is then isolated and purified by high pressure liquidchromatography.

As seen in FIG. 2, vitamin D₂ (11) is reacted with SO₂ and the hydroxylfunctionality at C-3 is protected with t-butyl dimethylsilylchlorideaffording the intermediate (12). Ozonolysis and reduction affords thealcohol (13). SO₂ extrusion, isomerization and subsequent oxidationusing the known Swern oxidation affords the aldehyde (14). The sidechain is introduced by reaction of aldehyde (14) with Wittig reagent toyield the 25-ene-vitamin D₂ compound (15). The 25-ene compound (15) isthen incubated with a cell line derived from human hepatoma cells, andthe 24-hydroxy-25-ene-vitamin D₂ (16) is extracted and purified.

The compounds of formula (III) may be generally prepared by theprocesses of FIGS. 1 and 2 wherein the previtamin starting materials canbe prepared by the exemplary reaction processes given in, e.g., U.S.Pat. No. 5,252,191 issued to Pauli et al.; U.S. Pat. No. 5,035,783issued to Goethals et al; U.S. Pat. No. 4,388,243, all of which areincorporated herein by reference. The 19-nor compounds of formula (1)may be generally prepared by the exemplary reaction processes givenherein wherein the 19-nor starting materials can be prepared by theexemplary processes given in, e.g., U.S. Pat. No. 5,710,294,incorporated herein by reference, using the appropriate vitamin Dstarting material.

The compounds of the present invention are useful as active compounds inpharmaceutical compositions having reduced side effects and low toxicityas compared with the known analogs of active forms of vitamin D₃. Thecompounds are especially of value for both local, including topical, andsystemic treatment and prophylaxis of human and veterinary disorderswhich are characterized by (i) abnormal cell proliferation and/or celldifferentiation, e.g., cancers such as skin, breast, colon and prostateand dermatological disorders such as psoriasis; (ii) imbalance of theimmune system, e.g., autoimmune diseases such as multiple sclerosis anddiabetes, and rejection of transplants; (iii) abnormal interleukin-1production, e.g., inflammatory response diseases such as rheumatoidarthritis and asthma; (iv) abnormal parathyroid hormone production,e.g., hyperparathyroidism, both primary and secondary; and (v) loss ofbone mass or bone mineral content, e.g., osteoporosis.

The 24-hydroxyvitamin D compounds of the present invention are thosethat have a lower tendency or inability to cause the undesired sideeffects of hypercalcemia and/or hypercalciuria. In other words, thecompounds of the present invention can be administered at dosages thatallow them to act, e.g., as antiproliferative agents and celldifferentiation agents when exposed to malignant or otherhyperproliferative cells, without significantly altering calciummetabolism. This selectivity and specificity of action makes the24-hydroxyvitamin D compounds of the present invention useful andpreferred agents for, e.g., safely inhibiting hyperproliferation andpromoting malignant or hyperplastic cell differentiation. The24-hydroxyvitamin D compounds of the present invention, thus, overcomethe shortcomings of the known active vitamin D₃ compounds describedabove, and can be considered preferred agents for the control andtreatment of malignant diseases such as prostate cancer as well asbenign prostatic hyperplasia, skin diseases, such as skin cancer andpsoriasis, breast cancer and colon cancer, immune and inflammatoryresponse disorders, and hyperparathyroidism as well as bone depletivedisorders.

The pharmacologically active compounds of this invention are suitablyprocessed in accordance with conventional methods of pharmacy to producemedicinal compositions for administration to patients, e.g., mammalsincluding humans, in, e.g., admixtures with conventional excipients suchas pharmaceutically acceptable organic or inorganic carrier substanceswhich do not deleteriously react with the active compounds, andoptionally, other therapeutic ingredients. Any suitable route ofadministration may be employed for providing an effective dosage of thecompounds in accordance with the present invention. For example, oral,rectal, topical, parenteral, intravenous, intramuscular, subcutaneous,ocular, nasal, buccal, and the like routes may be employed.

Therapeutic and prophylactic compositions are those suitable for thevarious routes of administration described herein, although the mostsuitable route in any given case will depend on the nature and severityof the condition being treated and on the nature of the activeingredient. The compositions are conveniently presented in unit dosageform.

Suitable pharmaceutically acceptable carriers for use in the compositionand method of the present invention include, but are not limited towater, salt solutions (e.g., buffer solutions), alcohols includingbenzyl alcohols, gum arabic, mineral and vegetable oils (e.g., corn oil,cottonseed oil, peanut oil, olive oil, coconut oil), fish liver oils,oily esters such as Polysorbate 80, polyethylene and propylene glycols,gelatin, carbohydrates (e.g., lactose, amylose or starch), magnesiumstearate, talc, silicic acid, viscous paraffin, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxymethylcellulose, polyvinyl pyrrolidone, etc.

The pharmaceutical preparations can be sterilized and, if desired, bemixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring and flavoring. If a solid carrier is used,the dosage form of the compounds of the present invention may betablets, capsules, powders, suppositories, or lozenges. If a liquidcarrier is used, soft gelatin capsules, transdermal patches, aerosolsprays, topical creams, syrups or liquid suspensions, emulsions orsolutions may be the dosage form.

It is noted, however, that dosage forms of 24-hydroxyprevitamin D aremost suitably formulated with carriers such as starch, lactose oramylose, which do not deleteriously react with the active compounds. Theformulations can be produced in tablet, capsule, powder, and lozengeform. However, whatever method of formulation is used, care should betaken to avoid exposure to solvents and heat as, under such conditions,there is a tendency for 24-hydroxyprevitamin D to convert to24-hydroxyvitamin D, i.e., the compounds of formula (III) are preferablyformulated in solvent-free, crystalline, heat-stable form. Because heatand solvents are to be avoided, the preferred method of tabletformulation is dry granulation.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Ampulesare convenient unit dosages. The dosage of the analogs in accordancewith the present invention for parenteral administration generally isabout 1–30 μg given 1 to 3 times per week.

As noted above, for enteral application, particularly suitable aretablets, dragées, liquids, drops, suppositories, or capsules. A syrup,elixir, or the like can also be used wherein a sweetened vehicle isemployed.

For rectal administration, compounds are formed into a pharmaceuticalcomposition containing a suppository base such as cacao oil or othertriglycerides. To prolong storage life, the composition advantageouslyincludes an antioxidant, such as ascorbic acid, butylated hydroxyanisoleor hydroquinone.

For topical application, there are also employed as nonsprayable forms,viscous to semi-solid or solid forms comprising a carrier compatiblewith topical application and having a dynamic viscosity preferablygreater than water. Suitable topical formulations include transdermaldevices, solutions, suspensions, emulsions, aerosols, creams, ointments,liniments, salves, lotions, dusting powders and the like which are, ifdesired, sterilized or mixed with auxiliary agents, e.g., preservatives,etc.

The magnitude of a prophylactic or therapeutic dose of the compounds inaccordance with the present invention will vary with the nature or theseverity of the condition to be treated and with the particularcomposition and its route of administration. Oral administration of thepharmaceutical compositions of the present invention is preferred.

In general, the daily dosage of the compounds according to thisinvention is about 0.025 to about 7.5 nmol/kg of body weight of thepatient, preferably about 0.025 to about 1 nmol/kg. The compounds ofthis invention are suitably dispensed by unit dosage form in apharmaceutically acceptable carrier, e.g., a unit dosage form includingabout 0.25 to about 50.0 μg in a pharmaceutically acceptable carrier perunit dosage. The dosage of the compounds according to the presentinvention generally is about 3.5 μg to about 1000 μg/week, preferablyabout 10 μg to about 500 μg/week.

For treatment of hyperproliferative diseases such as cancers andpsoriasis, the enteral dosage of the compounds of the present inventionis about 1 nmol to about 100 nmol per unit dosage; forhyperparathyroidism, about 0.5 nmol to 50 nmol per unit dosage; fortreatment of inflammatory diseases, about 1 nmol to 150 nmol per unitdosage; for immune response modulation, about 1 nmol to 150 nmol perunit dosage; and for bone depletive diseases, about 1 nmol to 150 nmolper unit dosage. In terms of micrograms, the effective dosage amount ona daily basis per kilogram of body weight of the patient ranges fromabout 0.01 μg/kg/day to about 3.0 μg/kg/day.

In addition, those skilled in the art will also appreciate that suchdosages may be encapsulated in time release, e.g., sustained, delayed ordirected release, delivery systems such as a liposome delivery system,polysaccharides exhibiting a slow release mechanism, salistic or otherpolymer implants or microspheres, as well as those where the activeingredient is suitably protected with one or more differentiallydegradable coatings, e.g., by microencapsulation, enteric coating,multiple coatings, etc., and such means effect continual dosing ofcompositions contained therein. For example, an enteric coating issuitably one which is resistant to disintegration in gastric juice. Itis also possible to freeze-dry the active ingredient and use thelyophilizate obtained, e.g., for the preparation of products forinjection.

It will be appreciated that the actual preferred amounts of activeanalog in a specific case will vary according to the specific compoundbeing utilized, the particular compositions formulated, the mode ofapplication, and the particular sites being treated. Dosages can bedetermined using conventional considerations, e.g., by customarycomparison of the differential activities of the subject compounds andof a known agent, e.g., by means of an appropriate conventionalpharmacological protocol.

The specific doses for each particular patient depend on a wide varietyof factors, for example, on the efficacy of the specific compoundemployed, on the age, body weight, general state of health, sex, on thediet, on the timing and mode of administration, on the rate ofexcretion, and on medicaments used in combination and the severity ofthe particular disorder to which the therapy is applied.

As described hereinbefore, the compounds of the present invention arepreferably administered to the human (or veterinary) patients in oraldosage formulation. As a compound in accordance with the presentinvention is released from the oral dosage formulation, it is absorbedfrom the intestine into the blood. The compounds of the presentinvention then undergo hydroxylation at the 1α-position of the A-ring ofthe vitamin D ring structure, thus providing an active form of thevitamin D compound which is 1α,24-dihydroxylated. As to the compounds offormula (I), little or no first-pass interaction with the intestinalvitamin D receptors is to be expected, thus, yielding little or nostimulation of intestinal calcium absorption. In the case of the24-hydroxyprevitamin D compounds of formula (III), as these compoundsare warmed by the core temperature of the animal or human, they convertto the corresponding 24-hydroxyvitamin D which are then 1α-hydroxylatedto form the 1,24-dihydroxy compounds. It is also noted that24-hydroxyprevitamin D compounds do not interact with the intestinalvitamin D receptors and, thus, do not stimulate first-pass intestinalcalcium absorption.

The dosage forms of the compositions of the present invention may alsocontain adjuvants as well as other therapeutically valuable substancesor may contain more than one of the compounds specified herein inadmixture. Thus, a further aspect within the scope of the presentinvention is administration of effective dosages of the compounds of thepresent invention in conjunction with administration of other hormonesor other agents which have been shown to have efficacy in the treatmentand prevention of the diseases and disorders described herein. It isanticipated that such co-administration or combination can provide asignificantly enhanced therapeutic effect, e.g., a synergistic effect.

For example, as to treatment of bone depletive diseases, compounds ofthe present invention are suitably co-administered with agents known toameliorate bone diseases or disorders. Such bone agents includeconjugated estrogens or their equivalents, antiestrogens, calcitonin,bisphosphonates, calcium supplements, calcium receptor agonists,cobalamin, pertussis toxin, boron, dehydroepiandrosterone (DHEA) andother bone growth factors such as transforming growth factor beta,activin or bone morphogenic protein. Possible dose ranges for certain ofthese co-administered agents are provided in Table 1.

TABLE 1 Possible Oral Dose Ranges for Various Agents Co-AdministeredWith 1α-Hydroxyvitamin D₂ Dose Ranges Most Agent Broad PreferredPreferred Conjugated Estrogens or 0.3–5.0 0.4–2.4 0.6–1.2 Equivalent(mg/day) Sodium Fluoride (mg/day) 5–150 30–75 40–60 Calcitonin (IU/day)5–800 25–500 50–200 Bisphosphonates (μg/day) 50–20,000 100–15,000250–10,000 Calcium Supplements (mg/day) 250–2500 500–1500 750–1000Calcium Receptor Agonists 4–1000 20–800 50–60 (mg/day) Cobalamin(μg/day) 5–200 20–100 30–50 Pertussis Toxin (mg/day) 0.1–2000 10–1500100–1000 Boron (mg/day) 0.10–3000 1–250 2–100 Antiestrogens, such asTamoxifen ™, are also known bone agents as well as antiproliferativeagents and may be suitably used in conjunction with the24-hydroxyvitamin D and 24-hydroxyprevitamin D compounds of the presentinvention.

Although the above examples detail dosage by mouth, it is to beunderstood that the combinations of agents can also be administered inalternative fashions, including intranasally, transdermally,intrarectally, intravaginally, subcutaneously, intravenously, andintramuscularly.

Also provided in the present invention is the co-administration of thecompounds of the present invention with known cytotoxic agents. Suchagents include estramustine phosphate, prednimustine, cisplatin,5-fluoro-uracil, melphalan, hydroxyurea, mitomycin, idarubicin,methotrexate, adriamycin and daunomycin. It is anticipated that avitamin D of formula (I) or (III) used in combination with variousanticancer drugs can give rise to a significantly enhanced cytotoxiceffect on cancerous cells, thus providing an increased therapeuticeffect. Specifically, as a significantly increased growth-inhibitoryeffect is obtained with the above-disclosed combinations utilizing lowerconcentrations of the anticancer drugs compared to the treatment regimesin which the drugs are used alone, there is the potential to providetherapy wherein adverse side effects associated with the anticancerdrugs are considerably reduced compared to those normally observed withthe anticancer drugs used alone in larger doses. Possible dose ranges ofthese co-administered second anticancer agents are about 0.1 to 1μg/kg/day.

The compounds in accordance with the present invention are also suitablyco-administered with known antiinflammatory agents. Such agents includeboth steroidal (e.g., corticosteroids) and nonsteriodal antiinflammatoryagents (e.g., salicylates, naproxen). It is anticipated that a compoundof the present invention used in combination with these variousantiinflammatory drugs can give rise to a significantly enhancedantiinflammatory activity, thus providing an increased therapeuticeffect and an anticipated lower effective dosage of antiinflammatoryagents.

Also included with the scope of the present invention is theco-administration of compounds in accordance with the present inventionwith known immune response augmenting agents. Such agents include thecyclosporins, DHEA and DHEA derivatives such as DHEA-sulfate,16α-bromo-DHEA, 7-oxo-DHEA, 16α-bromo-DHEA-sulfate and7-oxo-DHEA-sulfate. It is also anticipated that a compound of thepresent invention used in combination with these various immune responsemodulating drugs can give rise to a significantly enhancedimmunomodulating activity, thus providing an increased therapeuticeffect.

The present invention is further explained by the following exampleswhich should not be construed by way of limiting the scope of thepresent invention.

EXAMPLE 1 Synthesis of 24-hydroxyvitamin D₂ [24-OH-D₂]

(22E)-5,7,22-ergostatriene-3β-yl acetate (2)

To a solution of 50 gm (0.13 mol) of ergosterol (1) in 300 ml ofanhydrous pyridine was added 33.3 ml (0.35 mol) of acetic anhydride. Themixture was stirred at room temperature overnight and then 600 ml ofwater was added. The precipitate was filtered and washed three timeswith 200 ml portions of acetonitrile and then air dried to yield 42.0 g(74%) of (2).

22-oxo-5α,8α-(4-phenyl-3.5-dioxo-1,2,4-triazolidine-1.2-diyl)23,24-dinor-6-cholene-3β-ylacetate (4)

To a solution of 33.0 g (0.075 mol) of ergosterol acetate (2) in 1000 mlof chloroform was added 13.2 g (0.075 mol) of4-phenyl-1,2,4-triazoline-3,5-dione. The solution of the thus formed (3)was stirred at room temperature for 30 min. and then 5 ml of pyridinewas added. The solution was cooled to −78° C. and treated at −78° C.with an ozone-oxygen mixture for 2 hours and then thoroughly purged withnitrogen. Then 50 ml of dimethylsulfoxide was added and the mixture waswashed with 300 ml of water, then twice with 200 ml of 2N HC1 andfinally 300 ml of water. The organic layer was separated, dried overanhydrous MgSO₄ and concentrated to dryness in vacuo. The residue waspurified on a silica gel column using 30% ethyl acetate in hexane toyield 16.0 g (39%) of the title compound as a foamy solid.

¹H NMR: (400 MHZ; CDC1₃): δppm 0.85 (3H, s, 18-CH₃), 1.10 (3H, s,19-CH₃), 1.15 (3H, d, 21-CH₃), 1.99 (3H, s, 3β-CH₃CO), 5.45 (1H, m,3α-H), 6.26 (1H, d. 7-H), 6.40 (1H, d, 6-H), 7.42 (5H, m, Ph), 9.58 (1H,d, HCO).

(22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)cholesta-6,22-diene-24-one-3β-ylacetate (5)

Butyllithium (1.6 M solution in hexane 8.94 ml, 0.014 mol) was added toa stirred, cooled (0° C.) solution of diisopropylamine (1.45 g, 0.014mol) in dry tetrahydrofuran (20 ml) under nitrogen. 3-Methylbutan-2-one(1.23 g, 0.014 mol) in dry tetrahydrofuran (6 ml) was added dropwise at0° C. over 15 min. The solution was stirred at 0° C. for 1 hr. more,then cooled to −70° C. and a solution of the aldehyde (4) (6.0 g, 0.011mol) in dry tetrahydrofuran (60 ml) was added. The temperature wasraised to −20° C. and kept at this temperature for 3 hrs. Then glacialacetic acid (20 ml) was added at −20° C. and the solution was brought toroom temperature. Ether (800 ml) and water (400 ml) were added and theorganic layer was separated and washed with 10% hydrochloric acid (2×300ml), saturated sodium bicarbonate solution (2×300 ml), and water (2×300ml). Concentration gave the crude product (7.5 g) which was dissolved intetrahydrofuran (100 ml) containing 1.5 N-hydrochloric acid (12 ml).After refluxing for 1.5 hrs., the mixture was diluted with ether (600ml), washed with a 5% sodium carbonate solution (2×200 ml) and water(2×200 ml), and dried (anhydrous MgSO₄). Concentration under reducedpressure gave the crude product (7.0 g). Chromatography over silica gel(50% ethyl acetate in hexane) gave the enone (5) 4.0 g (59%).

¹H NMR: (400 MHZ): δppm 0.83 (3H, s. 18-CH₃), 0.99 (3H, s, 19-CH₃), 1.09(6H, dd, 26 and 27-CH₃), 1.12 (3H, d, 21-CH₃), 2.0 (3H, s, 3β-CH₃CO),2.84 (1H, m, 25-H), 5.45 (1H, m, 3α-H), 6.06 (1H, d, 23-H), 6.24 (1H, d,7-H), 6.39 (1H, d, 6-H), 6.71 (1H, dd, 22-H), 7.42 (5H, m, Ph).

(22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol(6)

The enone (5) (3.5 g, 5.7 mmol) in dry ether (100 ml) was cooled to 0°C. and methylmagnesium bromide (3.0 M solution in ether 6.8 ml, 0.02mol) was added dropwise. After 1 hr. at 0° C., saturated ammoniumchloride (100 ml) was added. The organic layer was separated. Theaqueous layer was extracted with ether (2×200 ml). The combined etherphases were dried over anhydrous MgSO₄ and concentrated to dryness invacuo to yield the crude product 3.0 g (90%) of (6).

(22E)-5,7,22-ergostatriene-3β,24-diol (7)

To a solution of 3.0 g (5.1 mmol) of (6) in dry tetrahydrofuran (250 ml)was added 3.6 g (0.09 mol) of lithium aluminum hydride. The mixture washeated under reflux for 3 hrs., cooled with ice water bath and reactionmixture decomposed by the cautious dropwise addition of ice water (5ml). The mixture was filtered and the filtrate was concentrated in vacuoto remove most of the tetrahydrofuran. The residue was dissolved in 200ml of ethyl acetate and washed twice with saturated NaCl solution (2×200ml), dried over anhydrous MgSO₄ and concentrated in vacuo. The residuewas purified on a silica gel column using 30% ethyl acetate in hexane toyield 1.5 g (71%) of (7).

¹H NMR: (400 MHZ, CDC1₃): δppm 0.64 (3H, s, 18-H), 0.88 (6H, dd, 26 and27-CH₃), 0.93 (3H, s, 19-CH₃), 1.06 (3H, d, 21-CH₃), 1.19 (3H, s,28-CH₃), 3.55 (1H, m, 3α-H), 5.36 (1H, d, 7-H), 5.42 (2H, m, 22 and23-H), 5.52 (1H, d, 6-H). UV (ethanol) λ_(max): 282 nm.

24-hydroxyvitamin D₂ (8)

One gram (2.4 mmol) of (7) was dissolved in 250 ml of ether and benzene(4:1) and irradiated with stirring under nitrogen in a water-cooledquartz immersion well using a Hanovia medium-pressure UV lamp for 2 hrs.The solution was concentrated in vacuo, redissolved in 100 ml of ethanoland heated under reflux overnight. The solution was concentrated todryness in vacuo and the residue was purified on a silica gel columnusing 30% ethyl acetate in hexane to yield 0.55 g (55%) of (8).

¹H NMR: (400 MHZ, CDC1₃): βppm 0.57 (3H, s, 18-CH₃), 0.92 (6H, dd, 26and 27-CH₃), 1.06 (3H, d, 21-CH₃), 1.20 (3H, s, 28-CH₃), 3.93 (1H, m,3-H), 4.79 (1H, m (sharp), 19-H), 5.01 (1H, m, (sharp), 19-H), 5.43 (2H,m, 22 and 23-H), 6.02 (1H, d, 7-H), 6.22 (1H, d, 6-H). UV (ethanol)λ_(max): 265 nm.

EXAMPLE 2 Synthesis of 24(S)-hydroxyvitamin D₂ (9) [24(S)-OH-D₂]

Product (8) is subjected to high pressure liquid chromatography (HPLC)on Zorbax-SIL developed with hexane/isopropanol/methanol (91:7:2) orusing a reverse-phase Supelco C-8 prep. column (25 cm×21.2 mm; particlesize 12 μm) with the solvent system, acetonitrile:water, 60:40, 10mL/min. The diastereomers (9) and (10) are eluted, and thereby,separated.

24(S)-Hydroxyvitamin D₂ was characterized as follows: optical rotation:[α]_(D) ^(24.0° C.)=+120.4 (c=1.0, ethanol); m.p.: 123–126° C.; TLC:R_(f)=0.10 (4:1, hexane, ethylacetate, silica; Whitman No. 4500-101),elemental analysis: calc'd. c=81.50, H=10.75; found c=81.62, H=10.66. Aninfrared spectrum (IR) (KBr) is shown in FIG. 3, and an nuclear magneticresonance (NMR) spectrum (300 MH₃, ¹H in CDC1₃) as given in FIG. 4.

EXAMPLE 3 In vivo generation of 1α,24(S)-dihydroxyvitamin D₂[1α,24(S)-(OH)₂-D₂] from 24(S)-OH-D₂

24-OH-D₂ was administered (either oral or intraperitonealsupplementation) to vitamin D-deficient rats. Lipid extracts of theplasma were prepared and the metabolites purified by the method of Horstet al. (Horst, R. L., Koszewski, N. J. and Reinhardt, T. A., Biochem.,29:578–82 (1990) and incorporated herein by reference) described belowfor synthesizing standard biological 1α,24-(OH)₂D₂.

Standard biological 1α,24-(OH)₂-D₂ was synthesized in vitro from24-OH-D₂ by incubating 10 μg of 24-OH-D₂ in flask containing 5 ml of 20%kidney homogenates made from vitamin D-deficient chicks. The product ofthis reaction was isolated by HPLC and identified by mass spectrometry.In the lipid extracts of the plasma from the vitamin D-deficient ratsadministered vitamin D₂ or 24-OH-D₂, one metabolite isolated co-migratedon HPLC with the standard 1α,24-(OH)₂D₂, indicating that 1α,24-(OH)₂D₂is a natural metabolite of vitamin D₂. In contrast, comparable ratsadministered vitamin D₃ had no detectable 24-OH-D₃.

EXAMPLE 4 In vivo generation of 1α,24(S)-(OH)₂-D₂ from24(S)-hydroxyprevitamin D₂ [24(S)-OH-previtamin D₂]

Male weanling rats are fed a diet deficient in vitamin D and with normalcalcium (0.47%). After a period of four weeks has elapsed, the rats aredivided into two groups, and orally administered either 24-OH-previtaminD₂ (0.25 μg/kg) in a vehicle such as lactose or the vehicle (control)alone. Four hours after administration, the rats are killed and theirblood level of 1α,24-(OH)₂-D₂ is measured using a standard technique.

Following this procedure demonstrates that the blood level of1α,24-(OH)₂-D₂ is significantly elevated over the blood level of controlanimals.

EXAMPLE 5 Production of 1α,24(S)-dihydroxy-25-ene-vitamin D₂[1α,24(S)-(OH)₂-25-ene-D₂] in osteoporotic women administered24-(OH)-25-ene-D₂

Human female subjects, who have been diagnosed with osteoporosis, aregiven daily doses of 25 μg/day 24-OH-25-ene-D₂ for one week. Blood iscollected and analyzed for the metabolite 1α,24(S)-(OH)₂-25-ene-D₂.Lipid is extracted from the blood, and the metabolite is purified byHPLC using standard methods and quantified with the radioreceptor assayproduced by Incstar (Stillwater, Minn.). One day following the last doseof 25 μg, the results show that there is a significant level of1α,24(S)-(OH)₂-25-ene-D₂ in the blood.

EXAMPLE 6 Treatment of osteoporosis with 24(S)-OH-preD₂

A clinical study is conducted with postmenopausal osteoporoticoutpatients having ages between 55 and 75 years. The study involves upto 120 patients randomly divided into three treatment groups, andcontinues for 24 months. Two of the treatment groups receive constantdosages of orally administered 24-OH-preD₂ (u.i.d.; two different doselevels above 5.0 μg/day) and the other group receives a matchingplacebo. All patients maintain a normal intake of dietary calcium (500to 800 mg/day) and refrain from using calcium supplements. Efficacy isevaluated by pretreatment and posttreatment comparisons of the patientgroups with regard to (a) total body, radial, femoral, and/or spinalbone mineral density as determined by x-ray absorptiometry (DEXA), (b)bone biopsies of the iliac crest, and (c) determinations of serumosteocalcin. Safety is evaluated by comparisons of urinaryhydroxyproline excretion, serum and urine calcium levels, creatinineclearance, blood urea nitrogen, and other routine determinations.

This study demonstrates that patients treated with orally administered24-OH-preD₂ exhibit significantly higher total body, radial, femoral,and/or spinal bone densities relative to patients treated with placebo.The treated patients also exhibit significant elevations in serumosteocalcin. Bone biopsies from the treated patients show that24-OH-preD₂ stimulates normal bone formation. The monitored safetyparameters confirm an insignificant incidence of hypercalcemia orhypercalciuria, or any other metabolic disturbance with 24-OH-preD₂.

EXAMPLE 7 Preventive treatment of bone mass loss in postmenopausalosteoporotic women using 24(S)-OH-25-ene-D₂

A clinical study is conducted with postmenopausal osteoporoticout-patients having ages between 55 and 75 years. The study involves upto 120 patients randomly divided into three treatment groups andcontinues for 24 to 36 months. Two of the treatment groups receiveconstant dosages of 24(S)-OH-25-ene-D₂ (u.i.d.; two different doselevels at or above 5.0 μg/day) and the other group receives a matchingplacebo. All patients maintain a normal intake of dietary calcium (500to 800 mg/day) and refrain from using calcium supplements. Efficacy isevaluated by pre- and post-treatment comparisons of the patient groupswith regard to (a) total body calcium retention, and (b) radial andspinal bone mineral density as determined by dual-photon absorptiometry(DPA) or dual-energy x-ray absorptiometry (DEXA). Safety is evaluated bycomparisons of urinary hydroxyproline excretion, serum and urine calciumlevels, creatinine clearance, blood urea nitrogen, and other routinedeterminations.

The results show that patients treated with 24(S)-OH-25-ene-D₂ exhibitsignificantly higher total body calcium, and radial and spinal bonedensities relative to patients treated with placebo. The monitoredsafety parameters confirm an insignificant incidence of hypercalcemia orhypercalciuria, or any other metabolic disturbance with24(S)-OH-25-ene-D₂ therapy.

EXAMPLE 8 Treatment of psoriasis with 24-OH-D₂

An oral dosage formulation containing 24-OH-D₂ is evaluated in a doubleblind study for therapeutic efficacy of the formulation in the treatmentof dermatitis (contact and ectopic). The formulation evaluated contains10.0 to 20.0 μg of 24-OH-D₂. The control formulation is identical exceptthat it does not contain the 24-OH-D₂. The patients are treated in anoutpatient clinic and are divided into an experimental and controlpopulation. They are instructed to take the medication once a day, inthe morning before breakfast.

In each patient (experimental and control) an area of the skincontaining a lesion is selected which is ordinarily covered by clothing,and the patients are instructed not to expose the skin area selected forstudy to sunlight. The area of the lesion is estimated and recorded, andthe lesion(s) is photographed. Relevant details of the photographicprocedure are recorded so as to be reproduced when the lesions are nextphotographed (distance, aperture, angle, background, etc.).

Evaluations of erythema, scaling, and thickness are conducted at weeklyintervals by a physician. The final evaluation is usually carried out atthe end of four to six weeks of treatment. The results of the study showthat daily oral administration of 24-OH-D₂ significantly reduces thedegree of erythema, scaling, and thickness versus the control patients.

EXAMPLE 9 Treatment of psoriasis with 24(S)-OH-preD₂

An oral dosage formulation containing 24(S)-OH-preD₂ is evaluated in adouble blind study for therapeutic efficacy of the formulation in thetreatment of dermatitis (contact and ectopic). The formulation evaluatedcontains 10.0 to 20.0 μg of 24(S)-OH-preD₂. The control formulation isidentical except that it does not contain the 24(S)-OH-preD₂. Thepatients are treated in an outpatient clinic and are divided into anexperimental and control population. They are instructed to take themedication once a day, in the morning before breakfast.

In each patient (experimental and control) an area of the skincontaining a lesion is selected which is ordinarily covered by clothing,and the patients are instructed not to expose the skin area selected forstudy to sunlight. The area of the lesion is estimated and recorded, andthe lesion(s) is photographed. Relevant details of the photographicprocedure are recorded so as to be reproduced when the lesions are nextphotographed (distance, aperture, angle, background, etc.).

Evaluations of erythema, scaling, and thickness are conducted at weeklyintervals by a physician. The final evaluation is usually carried out atthe end of four to six weeks of treatment. The results of the study showthat daily oral administration of 24(S)-OH-preD₂ significantly reducesthe degree of erythema, scaling, and thickness versus the controlpatients.

EXAMPLE 10 Treatment of prostate cancer using 24-(OH)-D₂

Patients with advanced androgen-independent prostate cancer participatein an open-label study of 24-(OH)-D₂. Qualified patients are at least 40years old, exhibit histologic evidence of adenocarcinoma of theprostate, and present with progressive disease which had previouslyresponded to hormonal intervention(s). On admission to the study,patients begin a course of therapy with oral 24-(OH)-D₂ lasting 26weeks, while discontinuing any previous use of calcium supplements,vitamin D supplements, and vitamin D hormone replacement therapies.During treatment, the patients are monitored at regular intervals for:(1) hypercalcemia, hyperphosphatemia, hypercalciuria, hyperphosphaturiaand other toxicity; (2) evidence of changes in the progression ofmetastatic disease; and (3) compliance with the prescribed test drugdosage.

The study is conducted in two phases. During the first phase, themaximal tolerated dosage (MTD) of daily oral 24-(OH)D₂ is determined byadministering progressively higher dosages to successive groups ofpatients. All doses are administered in the morning before breakfast.The first group of patients is treated with 25.0 μg of 24-(OH)D₂.Subsequent groups of patients are treated with 50.0, 75.0 and 100.0μg/day. Dosing is continued uninterrupted for the duration of the studyunless serum calcium exceeds 11.6 mg/dL, or other toxicity of grade 3 or4 is observed, in which case dosing is held in abeyance until resolutionof the observed toxic effect(s) and then resumed at a level which hasbeen decreased by 10.0 μg.

Results from the first phase of the study show that the MTD for24-(OH)D₂ is above 25.0 μg/day, a level which is 10- to 50-fold higherthan can be achieved with 1α,25-(OH)₂D₃. Analysis of blood samplescollected at regular intervals from the participating patients revealthat the levels of circulating 24-(OH)D₂ increase proportionately withthe dosage administered, rising to maximum levels well above 100 pg/mLat the highest dosages, and that circulating levels of 1α,25-(OH)₂D₃ aresuppressed, often to undetectable levels. Serum and urine calcium areelevated in a dose responsive manner. Patients treated with the MTD of24-(OH)D₂ for at least six months report that bone pain associated withmetastatic disease is significantly diminished.

During the second phase, patients are treated with 24-(OH)D₂ for 24months at 0.5 and 1.0 times the MTD. After one and two years oftreatment, CAT scans, X-rays and bone scans used for evaluating theprogression of metastatic disease show stable disease or partialremission in many patients treated at the lower dosage, and stabledisease and partial or complete remission in many patients treated atthe higher dosage.

EXAMPLE 11 Treatment of prostate cancer using 24-OH-preD₂

The study of Example 10 is repeated for the vitamin D compound,24-OH-preD₂. The results of the phase one study indicate that patientstreated with the MTD of 24-OH-preD₂ for at least six months report thatbone pain associated with metastatic disease is significantlydiminished. The results of the phase two study indicate that after twoyears, CAT scans, X-rays and bone scans used for evaluating theprogression of metastatic disease show stable disease or partialremission in many patients treated at the lower dosage, and stabledisease and partial or complete remission in many patients treated atthe higher dosage.

EXAMPLE 12 Treatment of elderly subjects with elevated blood PTH fromsecondary hyperparathyroidism with 24-OH-D₄

A twelve-month double-blind placebo-controlled clinical trial isconducted with forty subjects with secondary hyperparathyroidism. Theselected subjects have ages between 60 and 100 years and have a historyof secondary hyperparathyroidism. Subjects also have femoral neckosteopenia (femoral neck bone mineral density of ≦0.70 g/cm²).

All subjects enter a six-week control period after which the subjectsare randomized into two treatment groups: one group receives a constantdosage of 15 μg/day 24-(OH)-D₄, and the other group receives a matchingplacebo. Both groups maintain a normal intake of dietary calcium withoutthe use of calcium supplements. Efficacy is evaluated by pre- andpost-treatment comparisons of the two patient groups with regard to (a)intact PTH (iPTH); (b) radial, femoral and spinal bone mineral density;and (c) bone-specific urine markers (e.g., pyridinium crosslinks).Safety is evaluated by (a) serium calcium and phosphorus, and (b) urinecalcium and phosphorus.

Analysis of the clinical data show that 24-(OH)-D₄ significantlydecreases iPTH and bone specific urine markers. Subjects treated withthis compound show normal serum calcium levels and stable radial andspinal bone densities relative to baseline values. In contrast, patientstreated with placebo show no reduction in iPTH and bone-specific urinemarkers. An insignificant incidence of hypercalcemia is observed in thetreatment group.

EXAMPLE 13 Treatment of elderly subjects with elevated blood PTH fromsecondary hyperparathyroidism with 24-OH-preD₂

A twelve-month double-blind placebo-controlled clinical trial isconducted with forty subjects with secondary hyperparathyroidism. Theselected subjects have ages between 60 and 100 years and have a historyof secondary hyperparathyroidism. Subjects also have femoral neckosteopenia (femoral neck bone mineral density of ≦0.70 g/cm²).

All subjects enter a six-week control period after which the subjectsare randomized into two treatment groups: one group receives a constantdosage of 15 μg/day 24-OH-preD₂, and the other group receives a matchingplacebo. Both groups maintain a normal intake of dietary calcium withoutthe use of calcium supplements. Efficacy is evaluated by pre- andpost-treatment comparisons of the two patient groups with regard to (a)intact PTH (iPTH); (b) radial, femoral and spinal bone mineral density;and (c) bone-specific urine markers (e.g., pyridinium crosslinks).Safety is evaluated by (a) serium calcium and phosphorus, and (b) urinecalcium and phosphorus.

Analysis of the clinical data show that 24-OH-preD₂ significantlydecreases iPTH and bone specific urine markers. Subjects treated withthis compound show normal serum calcium levels and stable radial andspinal bone densities relative to baseline values. In contrast, patientstreated with placebo show no reduction in iPTH and bone-specific urinemarkers. An insignificant incidence of hypercalcemia is observed in thetreatment group.

EXAMPLE 14 Treatment of patients with secondary hyperparathyroidism inend stage renal disease using 24-OH-D₂

Thirty renal patients are enrolled in a clinical trial to studysecondary hyperparathyroidism. The patients show baseline iPTH levelsgreater than 1000 pg/mL. An initial dose of 24-OH-D₂ (50 μg 3times/week) is increased (maximum, 100 μg 3 times/ week) or decreased asnecessary to attain and maintain iPTH in the range of 150–300 pg/mL.After 11–12 weeks of treatment, the iPTH levels of the patients decreaseto below 1000 pg/mL, and in many cases to below 500 pg/mL. There are fewepisodes of hypercalcemia with the patients during the study.

EXAMPLE 15 Treatment of primary hyperparathyroidism with 24-OH-preD₂

Twenty renal patients are enrolled in a clinical trial to study primaryhyperparathyroidism. The patients show baseline iPTH levels greater than200 pg/mL. An initial dose of 24-OH-preD₂ (2–4 μg/day) is increased(maximum, 10 μg/day) or decreased as necessary to attain and maintainiPTH in the normal range. After 11–12 weeks of treatment, the iPTHlevels of the patients decrease to below 100 pg/mL, and in many cases tobelow 60 pg/mL. There are few episodes of hypercalcemia with thepatients during the study.

EXAMPLE 16 Immunological testing of 24-OH-D₂

Female C57BL/6 mice are used between the ages of 9–12 weeks. Mice aregiven food and water ad libitum and are kept in a 12-hour light and12-hour dark cycle.

A known balanced salt solution (BSS) is prepared and supplemented to0.01 molar with HEPES buffer.

The test compound, 24-OH-D₂, is dissolved in dimethylsulfoxide at finalconcentrations of 0.2 or 0.4 mg per ml. When working with vitamin Dcompounds, conditions of reduced lighting were employed.

Mice are apportioned at 4 per group and are inoculated intraperitoneallywith 3×10⁶ allogeneic P815 tumor cells and the resulting cytotoxicthymus-derived lymphocyte (CTL) activity is assessed 10 days later. Miceare treated by the intraperitoneal route with 25 microliters of testcompound dissolved in dimethylsulfoxide or with dimethylsulfoxide only(vehicle control). In test 1, mice are given daily treatments of 5micrograms of 24-OH-D₂ per day starting one day before immunization andcontinuing until the day before assay. In test 2, mice are treated with10 micrograms of 24-OH-D₂ only twice: on the day before immunization andon the day of immunization.

Ten days after immunization of mice with P815 cells, single spleen cellsuspensions are prepared by passage of spleens through a steel mesh intoBSS and are subsequently washed twice with BSS. Further manipulations ofspleen cells, labeling of P815 target cells with Cr, mechanics of theassay, and the calculation of results from the CTL assay are known anddescribed in U.S. Pat. No. 4,749,710, incorporated herein by reference.Cytotoxic T lymphocyte activity is determined individually on spleencells from each animal in each group and the results are expressed asthe mean CTL activity (as percent specific Cr release) of each group ±the standard deviation.

The results show that mice immunized with P815 cells developedsubstantial CTL activity within 10 days in the vehicle control groups. Astatistically significant reduction in CTL activity is seen in bothtests in those groups which were treated with 24-OH-D₂, thus documentingthe immunosuppressive activity of the compound when administered toanimals.

It is to be understood that although the foregoing examples detail useof specific 24-OH-D and 24-OH-preD compounds, other compounds within thescope of the claims may be readily utilized in the treatment methods ofthe present invention with essentially equivalent results.

In summary, the present invention provides 24-hydroxyvitamin D prodrugcompounds which include 24-hydroxyprevitamin D compounds that in vivoare 1α-hydroxylated to 1,24-hydroxyvitamin D compounds. The compoundsare particularly adapted to elicit significantly less hypercalcemia thandosing with similar amounts of vitamin D₃ compounds such as1,25-dihydroxy vitamin D₃. The compounds are useful in the treatment andprevention of hyperparathyroidism, hyperproliferative diseases as wellas bone depletive disorders, and immunological and inflammatory responseregulation.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions, and omissions, that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation that lawfully can be accorded the appendedclaims.

1. A method of achieving an effect in a patient in need thereofcomprising administering an effective amount of a vitamin D compoundwhich is a 24-hydroxyvitamin D compound wherein the effect is treatinghyperparathyroidism, and wherein said 24-hydroxyvitamin D is a compoundof formula (I):

wherein Z is a side chain of formula (IIB):

wherein R⁵ and R⁶ are each hydrogen or taken together form a double bondbetween C-22 and C-23, R³ is hydrogen, lower alkyl, lower fluoroalkyl,lower alkenyl or lower fluoroalkenyl; R⁴ is lower alkyl, lowerfluoroalkyl, lower alkenyl or lower fluoroalkenyl; and R¹ and R² areindependently hydrogen, lower alkyl, lower fluoroalkyl, lower alkenyl,lower fluoroalkenyl, lower cycloalkyl or taken together with the carbonto which they are bonded form a C₃–C₈ cyclocarbon ring; Y is a methylenegroup if Y is double bonded to the A-ring or a methyl group or hydrogenif Y is single bonded; and X is hydrogen, lower alkyl or lowerfluoroalkyl.
 2. A method of achieving an effect in a patient in needthereof comprising administering an effective amount of a vitamin Dcompound which is a 24-hydroxyprevitamin D, wherein the effect istreating hyperparathyroidism, wherein said 24-hydroxyprevitamin D is acompound of formula (III):

wherein Z is a side chain of formula IIB, IID or IIF:

wherein R⁵ and R⁶ are each hydrogen or taken together form a double bondbetween C-22 and C-23, R³, R⁹ and R¹⁰ are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl or lower fluoroalkenyl; R⁴ andR⁷ are independently lower alkyl, lower fluoroalkyl, lower alkenyl orlower fluoroalkenyl; and R¹ and R² are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl, lower fluoroalkenyl, lowercycloalkyl or taken together with the carbon to which they are bondedform a C₃–C₈ cyclocarbon ring; Y is a methyl group or hydrogen; and X ishydrogen, lower alkyl or lower fluoroalkyl.
 3. A method of achieving aneffect in a patient in need thereof comprising administering aneffective amount of a vitamin D compound which is a 24-hydroxyvitamin D,wherein the effect is lowering or maintaining lowered parathyroidhormone levels, and wherein the 24-hydroxyvitamin D is a compound offormula (I):

wherein Z is a side chain of formula IIB, IID or IIF:

wherein R⁵ and R⁶ are each hydrogen or taken together form a double bondbetween C-22 and C-23, R³, R⁹ and R¹⁰ are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl or lower fluoroalkenyl; R⁴ andR⁷ are independently lower alkyl, lower fluoroalkyl, lower alkenyl orlower fluoroalkenyl; and R¹ and R² are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl, lower fluoroalkenyl, lowercycloalkyl or taken together with the carbon to which they are bondedform a C₃–C₈ cyclocarbon ring; Y is a methylene group if Y is doublebonded to the A-ring or a methyl group or hydrogen if Y is singlebonded; and X is hydrogen, lower alkyl or lower fluoroalkyl.
 4. A methodof achieving an effect in a patient in need thereof comprisingadministering an effective amount of a vitamin D compound which is a24-hydroxyprevitamin D wherein the effect is lowering or maintaininglowered parathyroid hormone levels, wherein said 24-hydroxyprevitamin Dis a compound of formula (III):

wherein Z is a side chain of formula IIB, IID or IIF:

wherein R⁵ and R⁶ are each hydrogen or taken together form a double bondbetween C-22 and C-23, R³, R⁹ and R¹⁰ are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl or lower fluoroalkenyl; R⁴ andR⁷ are independently lower alkyl, lower fluoroalkyl, lower alkenyl orlower fluoroalkenyl; and R¹ and R² are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl, lower fluoroalkenyl, lowercycloalkyl or taken together with the carbon to which they are bondedform a C₃–C₈ cyclocarbon ring; Y is a methyl group or hydrogen; and X ishydrogen, lower alkyl or lower fluoroalkyl.
 5. A method of treating ahuman in need thereof to alleviate the pathological effects ofhyperparathyroidism, wherein the method comprises administering to thehuman in need thereof a vitamin D compound which is a 24-hydroxyvitaminD of formula (I), wherein said compound is administered to the human inneed thereof in an amount sufficient to lower or maintain loweredparathyroid hormone level in the human in need thereof; wherein formula(I) is:

wherein Z is a side chain of formula IIB, IID or IIF:

wherein R⁵ and R⁶ are each hydrogen or taken together form a double bondbetween C-22 and C-23, R³, R⁹ and R¹⁰ are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl or lower fluoroalkenyl; R⁴ andR⁷ are independently lower alkyl, lower fluoroalkyl, lower alkenyl orlower fluoroalkenyl; and R¹ and R² are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl, lower fluoroalkenyl, lowercycloalkyl or taken together with the carbon to which they are bondedform a C₃–C₈ cyclocarbon ring; Y is a methylene group if Y is doublebonded to the A-ring or a methyl group or hydrogen if Y is singlebonded; and X is hydrogen, lower alkyl or lower fluoroalkyl.
 6. Themethod of claim 1, wherein said 24-hydroxyvitamin D compound isadministered in a dosage of about 3.5 μg to about 1000 μg/week.
 7. Themethod of claim 2 wherein Z is a side chain of formula (IIB).
 8. Themethod of claim 7, wherein said 24-hydroxyprevitamin D is24-hydroxyprevitamin D₂; 24(S)-hydroxyprevitamin D₂;24-hydroxyprevitamin D₄; or 24(R)-hydroxyprevitamin D₄.
 9. The method ofclaim 2 wherein Z is a side chain of formula (IID).
 10. The method ofclaim 9 wherein said 24-hydroxyprevitamin D compound is24-OH-25-ene-preD₂.
 11. The method of claim 1, wherein the effect istreating hyperparathyroidism.
 12. The method of claim 1, wherein thevitamin D compound is 24-hydroxy-19-nor-vitamin D.
 13. The method ofclaim 1, wherein the vitamin D compound is 24-hydroxyvitamin D₂.
 14. Themethod of claim 2, wherein the effect is treating hyperparathyroidism.15. The method of claim 2, wherein the vitamin D compound is24-hydroxyprevitamin D₂.
 16. The method of claim 3, wherein the effectis lowering or maintaining lowered parathyroid hormone level.
 17. Themethod of claim 3, wherein the vitamin D compound is24-hydroxy-19-nor-vitamin D.
 18. The method of claim 3, wherein thevitamin D compound is 24-hydroxyvitamin D₂.
 19. The method of claim 4wherein the effect is lowering or maintaining lowered parathyroidhormone level.
 20. The method of claim 4, wherein the vitamin D compoundis 24-hydroxyprevitamin D₂.
 21. The method of claim 5 wherein the methodof treatment alleviates the pathological effects of hyperparathyroidism.22. The method of claim 5 wherein the vitamin D compound is administeredto the human in an amount sufficient to lower or maintain loweredparathyroid hormone level.
 23. The method of claim 5, wherein thevitamin D compound is 24-hydroxy-19-nor-vitamin D.
 24. The method ofclaim 5, wherein the vitamin D compound is 24-hydroxyvitamin D₂.
 25. Themethod of claim 5 wherein the Z side chain is formula IIB.
 26. Themethod of claim 25, wherein said 24-hydroxyvitamin D is24-hydroxyvitamin D₂; 24(S)-hydroxyvitamin D₂; 24-hydroxyvitamin D₄;24(R)-hydroxyvitamin D₄.
 27. The method of claim 5 wherein the Z sidechain is formula IID.
 28. The method of claim 5 wherein the Z side chainis formula IIF.
 29. A method of treating a human in need thereof toalleviate the pathological effects of hyperparathyroidism, wherein themethod comprises administering to the human in need thereof a vitamin Dcompound which is a 24-hydroxyprevitamin D of formula (III), whereinsaid compound is administered to the human in need thereof in an amountsufficient to lower elevated or maintain lowered parathyroid hormonelevel in the human in need thereof; wherein formula (III) is:

wherein Z is a side chain of formula IIB, IID or IIF:

wherein R⁵ and R⁶ are each hydrogen or taken together form a double bondbetween C-22 and C-23, R³, R⁹ and R¹⁰ are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl or lower fluoroalkenyl; R⁴ andR⁷ are independently lower alkyl, lower fluoroalkyl, lower alkenyl orlower fluoroalkenyl; and R¹ and R² are independently hydrogen, loweralkyl, lower fluoroalkyl, lower alkenyl, lower fluoroalkenyl, lowercycloalkyl or taken together with the carbon to which they are bondedform a C₃–C₈ cyclocarbon ring; Y is a methyl group or hydrogen; and X ishydrogen, lower alkyl or lower fluoroalkyl.
 30. The method of claim 29wherein the method of treatment alleviates the pathological effects ofhyperparathyroidism.
 31. The method of claim 29 wherein the Z side chainis formula IIB.
 32. The method of claim 29, wherein said24-hydroxyprevitamin D is 24-hydroxyprevitamin D₂;24(S)-hydroxyprevitamin D₂; 24-hydroxyprevitamin D₄;24(R)-hydroxyprevitamin D₄.
 33. The method of claim 32, wherein thevitamin D compound is 24-hydroxyprevitamin D₂.
 34. The method of claim29 wherein the Z side chain is formula IID.
 35. The method of claim 29wherein the Z side chain is formula IIF.